Over the past two decades numerous investigators have devoted significant effort to the study of partially purified extracts of mammalian tissue and fluids in order to identify and confirm the existence of endogenous humoral factors that may be involved in the regulation of cation transport in tissues as well as in the regulation of sodium excretion. At present, considerable evidence has been produced supporting the existence of such an endogenous factor or family of factors that is believed to inhibit the Na.sup.+, K.sup.+ -ATPase enzyme system which regulates active sodium transport. Moreover, these inhibitory properties implicate the involvement of such factors in several physiological roles including natriuresis, the excretion of abnormal amounts of sodium in the urine, as well as in the genesis of certain forms of hypertension and cardiac malfunction.
However, in spite of the extensive data produced by these early investigators, considerable controversy exists with respect to the identity and characterization of such endogenous factors as well as with respect to their mechanisms of action. It is believed that the primary reason for this controversy may be due to the fact that these early investigators often obtained conflicting and contradictory results due to their inability to effectively purify the tissue and fluid extracts utilized in their studies. A consequence of this reported disparity in described characteristics has been to suggest that the inhibitory factors identified and studied in the prior art may be of more than a single type or, possibly, may be a variety of relatively low potency substances producing non-specific inhibition.
The early research in this field initially suggested that an endogenous humoral factor, or factors, may have been involved in regulating cation transport in vital tissues as well as in the regulation of sodium excretion by the kidney. Such factors were also indicated as being active in regulating vascular reactivity in arterioles. More specifically, these early studies demonstrated in dogs that an infusion of saline produced a rise in urinary sodium excretion even when the glomerular filtration rate and renal blood flow were reduced. See, de Wardener, H. E., et al., "Studies On The Efferent Mechanism Of The Sodium Diuresis Witch Follows The Administration Of Intravenous Saline In The Dog." Clin Sci. 1961;21:249-258. That a humoral factor was involved in regulating sodium transport was also indicated by the research of Dahl, L. K., et al., "Humoral Transmission Of Hypertension: Evidence From Parabiosis." Cir Res. 1969;24 (suppl 1): 21-23 where a salt-resistant rat was placed in parabiosis with a salt-sensitive rat and hypertension developed in the resistant rat when both animals were fed salt. Thus, under these conditions, it was presumed that a humoral hypertensive agent passed from the salt-sensitive rat to the salt-resistant rat.
Later research confirmed these preliminary results and also demonstrated that plasma extract from saline-loaded and volume-expanded dogs exhibited natriuretic, pressor, vascular sensitizing, and digoxin-like activities. See, Knock, C. A., et al. "Evidence In Vivo For A Circulating Natriuretic Substance In Rats After Expanding The Blood Volume." Clin Sci. 1980;59:411-421; Sonnenburg, H., et al., "A Humoral Component Of The Natriuretic Mechanism In Sustained Blood Volume Expansion." J Clin Invest. 1972;51:1631-1634 Knock, C. A., "Further Evidence In Vivo For A Circulating Natriuretic Substance After Expanding The Blood Volume In Rats." Clin Sci. 1980;59:423433; Pearce, J. W., et al., "Time Course Of Onset And Decay On Humoral Natriuretic Activity In The Rat." Can J Physiol Pharmacol. 1975;53:734-741; Bergele, A. H., et al., "Development Of Renal Response To Blood Volume Expansion In the Rat." Am J Physiol. 1974;227:364-368; Krecek, J., et al., "Sensitivity Of Prepubertal Rats To The Hypertensiogenic Effect Of Salt-A Lack Of Natriuretic Factor." In: Lichardus, B., et al., "Hormonal Regulation Of Sodium Excretion." Amsterdam: Elsevier North-Holland; 1980:289-297; Haddy, F. J., et al., "The Role Of Humoral Factors In Volume Expanded Hypertension." Life Sci. 1976;19:935-948; Gonick, H. C., et al., "Circulating Inhibitor Of Sodium-Potassium Activated Adenosine Triphosphatase After Expansion Of Extracellular Fluid Volume Rats." Clin Sci Mol Med. 1977;53:329-334.
Subsequent research provided evidence that a common humoral factor may be responsible for inhibition of Na.sup.+, K.sup.+ -ATPase and sodium transport. For example, numerous studies have supported the proposition that the inhibition of Na.sup.+, K.sup.+ -ATPase in the smooth muscle of the arteriole increases intracellular sodium, which increases intracellular calcium concentration and, as a result, the arteriolar tone. See, Blaustein, M. P., "Sodium, Ions, Calcium Ions, Blood Pressure Regulation And Hypertension: A Reassessment And A Hypothesis." Am J Physiol. 1977;232:C165-C173; Overbeck, H. W., et al., "Depressed Function Of A Ouabain-Sensitive Sodium-Potassium Pump In Blood Vessels From Renal Hypertensive Dogs." Circ Res. 1976;38(suppl 2):48-52; Pamnani, M., et al., "Demonstration Of A Humoral Inhibitor Of The (Na,K) Pump In Some Models Of Experimental Hypertension." Hypertension 1981;3,No. 6.:96-101; Pamnani, J. B., et al., "Vascular Sodium-Potassium Pump Activity In Various Models Of Experimental Hypertension." Clin Sci. 1980;59:-179 S-181 S; Hamlyn, J. M., et al. "A Circulating Inhibitor Of (Na,K)ATPase Associated With Essential Hypertension." Nature. 1982;300:650652; Poston, L., et al., "Evidence For A Circulating Sodium Transport Inhibitor In Essential Hypertension." Br Med J. 1981;282:1267-1269; MacGregor, G. A., et al., "Evidence For A Raised Concentration Of A Circulating Sodium Transport Inhibitor In Essential Hypertension." Br Med J. 1981;283:1355-1357; Devynck, M. A., et al., "Circulating Digitalis-Like Compounds In Essential Hypertension." Clin Exp Hypert. 1984;6:441-453.
Moreover, these studies also indicated that the factor or factors possessing these properties could function as an endogenous diuretic as well as be involved in certain forms of hypertension. For example, Poston, et al. demonstrated that normal leukocytes had low active sodium eflux and high intracellular sodium concentrations following incubation with the plasma of patients having essential hypertension. Similarly, Hamlyn, et al., MacGregor, et al., and Devynck, et al. showed that plasma from hypertensive patients contained a factor or factors that inhibited Na.sup.+, K.sup.+ -ATPase.
Additional studies also supported the clear implication that such inhibitory factors are involved in the pathogenesis of hypertension. For example, the presence of a digoxin-like immunoreactive substance was demonstrated in the amniotic fluid of hypertensive pregnant women and in the plasma of preeclamptic pregnant patients. See. Graves, S. W., et al., "An Endogenous Ouabain-Like Factor Associated With Hypertensive Pregnant Women." J Clin Endocr Matab. 1984;59:1070-1075; Gudson, J. O., et al. "A Digoxin-Like Immunoreactive Substance In Preeclampsia." Am J Obstet Gynecol. 1984;150:83-85. However, as with the previously discussed research, the identity and characterization of the factor or factors in question remained obscure.
More recent research has been directed at attempting to identify the source of these suspected inhibitory factors. Several investigators have attempted to isolate such factors from brain extract and digitalis-like activity was reported in brain extract after various purification steps. See, Fishman, M.D., "Endogenous Digitalis-Like Activity In Mammalian Brain." Proc Natl Acad Sci. 1979;76.No.9.:4661-4663; Lichtstein, D., et al. "Endogenous Ouabain-Like Activity In Rat Brain." Biochem Biophys Res Comm. 1980;96,No.4:1518-1523. Similarly, it has been reported that a lesion in the anterioventral third ventricle in the volume-expanded rat decreased the level of Na.sup.+, K.sup.+ -ATPase inhibition. See, Pamnani, M., et al. "Demonstration Of A Humoral Inhibitor Of The (Na,K) Pump In Some Models Of Experimental Hypertension." Hypertension 1981;3,No.6.:96-101; Pamnani, M., et al., "Vascular Na.sup.+ -K.sup.+ Pump Activity In 'Acutely Saline-Loaded Rats With Anterioventral Third Ventricle (AV3V) Lesions [Abstract]." Fed Proc. 1981;40-390.
Additional research has focussed on more specific brain tissue sources. For example, extracts of bovine hypothalamus were reported by the present inventor to contain a factor that reduced sodium transport across anural membranes and also inhibited canine renal Na.sup.+, K.sup.+ -ATPase. See, Haupert G. T., et al. "Sodium Transport Inhibitor From Bovine Hypothalamus." Proc Natl Acad Sci. 1979;76:4658-4660. However, these extracts were only partially purified and failed to characterize or identify the specific factor. Inhibitory activity has also been reported in extracts of rat hypothalamus and in cultures of hypothalamic neurons as well as from bovine adrenal tissues. See, Alaghband-Zadeh, et al., "Evidence That The Hypothalamus May Be A Source Of A Circulating Na.sup.+ -K.sup.+ -ATPase Inhibitor." J Endocrinol. 1983;98:221-226; Morgan, K., et al., "Release Of An Active Sodium Transport Inhibitor (ASTI) From Rat Hypothalamic Cells In Culture." Endocrinol. 1984;115,No.4:1642-1644; Tamura, M., et al. "Isolation and Characterization Of A Specific Endogenous Na.sup.+,K.sup.+ -ATPase Inhibitor From Bovine Adrenal." Biochemistry. 988;27:4244-4253.
Other tissue and fluid extracts have also been examined. Plasma and urine extracts from volume expanded animals and humans have been reported to exhibit a natriuretic effect in over 25 separate studies as reviewed by de Wardener and Clarkson. See, de Wardener, H. E., et al., "Concept Of Natriuretic Hormone." Physiol Rev. 1985;65,No.3:658-759. Additionally, three fractions have been extracted from normal plasma which exhibit Na.sup.+, K.sup.+ -ATPase inhibitory activity. See, Kelly, R. A., et al., "Characterization Of Digitalis-Like Factors In Human Plasma." J Biol Chem. 1985;260 no.21:11396-11405. These fractions are also reported to cross react with digoxin antibodies.
Similar digoxin-like activity, such as cross-reaction with anti-digoxin antibodies, has been reported in deproteinized normal serum and urine. See. Balzan, S., et al., "Digoxin-Like Immunoreactivity In Normal Human Plasma And Urine, As Detected By A Solid-Phase Radioimmunoassay." Clin Chem. 1984;30:450-451; Cloix, J. F., et al. "High Yield-Purification Of A Urinary Na-Pump Inhibitor." Biochem Biophys Res Comm. 1985;131:1234-1240. Cloix, et al. believe that their digoxin-like sodium transport inhibitor is an amino-glyco steroid.
One group of investigators has partially isolated a sodium pump inhibitor from a variety of animal tissues utilizing reverse phase HPLC. See, Fagoo, M., et al., "Further Characterization Of Cardiodigin,Na.sup.+ -K.sup.+ -ATPase Inhibitor Extracted From Mammalian Tissues." Febs Letters. 1985;184:1631-1634. Evidencing some of the confusion regarding the characterization of such compounds, they have designated this inhibitor "cardiodigin" and believe it to be one of the recently identified lignan compounds found in mammalian fluids. Lignans are known to have several structural similarities to cardiac glycosides including a gamma butyrolactone ring which is deemed necessary for interaction with Na.sup.+, K.sup.+ -ATPase. See, Fagoo, M., et al., "Evidence That Mammalian Lignans Show Endogenous Digitalis-Like Activities." Biochim Biophys Acta. 1986;134 n.4:1064-1070.
Further contributing to the confusion regarding the identify of these compounds, other endogenous molecules have also been implicated in Na.sup.+, K.sup.+ -ATPase inhibition. These include the free fatty acids and ascorbic acid. Like the lignans, ascorbic acid also possesses a gamma butyrolactone ring and inhibits Na.sup.+, K.sup.+ -ATPase. See. Ng, I., et al. "Ascorbic Acid: An Endogenous Inhibitor Of Isolated Na.sup.+ -K.sup.+ -ATPase." Biochem Pharmacol. 1985;34:2525-2530. However, ascorbic acid does not affect sodium transport in cells and therefore may not be a physiological regulator of sodium transport. Conversely, the free fatty acids have been implicated as endogenous regulators of sodium transport and have also
been demonstrated to inhibit Na.sup.+, K.sup.+ -ATPase. See, Bidard, J. N., et al. "A Search For An Ouabain-Like Substance From The Electric Organ Of Electrophorus Electricus With Led to Arachidonic Acid and Related Fatty Acids." Biochem Biophys Res Comm. 1984;-Actas 769:245-252; Tamura, M., et al. "Identification Of Linoleic And Oleic Acids As Endogenous Na.sup.+ -K.sup.+ -ATPase Inhibitors From Acute Volume-expanded Hog Plasma. (Abs.)." J Biol Chem. 1985;260:9672-9677. In this research by Tamura, et al. it was shown that the plasma of acutely volume-expanded pigs exhibited inhibitory activity and digoxin-like activity. Most of the reported activity was accounted for by an increase in linoleic acid and oleic acid, suggesting that free fatty acids regulate sodium excretion in acute volume expansion through depression of tubular sodium reabsorption. However, involvement of free fatty acids in the regulation of sodium excretion in normal physiological circumstances remains unknown.
As evidenced by the foregoing, in spite of the extensive research in this field, little agreement exists among the various investigators regarding the origin and character of such sodium transport inhibitory factors. In addition to the free fatty acids, lignans and other compounds previously discussed, some investigators have suggested that the factor or factors may be steroidal in view of the many similarities between the endogenous sodium transport inhibitor and digitalis. See. Cloix, et al. "High Yield-Purification Of A Urinary Na-Pump Inhibitor." Biochem Biophys Res Comm 1985;131:1234-1240; LaBella, F. S., et al. "Progesterone Derivatives That Bind To The Digitalis Receptor: Effects On Na.sup.+ -K.sup.+ -ATPase And Isolated Tissues." Fed Proc. 1985;44:2806-2828.
Conversely, other investigators have also identified the factor as a peptide. See, Kramer, H. J., et al. "Further Characterization Of The Endogenous Natriuretic And Digoxin-Like Immunoreacting Activities In Human Urine: Effects Of Changes In Sodium Intake." Renal Physiol. 1985;8:80-89. Similarly, two of the investigators using the hypothalamus as their tissue source have also presented evidence that the extracted factor or factors is a peptide. See. Morgan, K., et al. "Characterization And Partial Purification Of the (Na,K)ATPase Inhibitor Released From Cultured Rat Hypothalamic Cells." J Bio Chem. 1985;25:13595-13600; Akagawa, K., et al. "Partial Purification And Properties Of The Inhibitors Of Na,K-ATPase And Ouabain-binding In Bovine Central Nervous System." J Neurochem. 1984;42:775-780.
However, other researchers utilizing hypothalamic tissue sources have identified partially purified factor extracts which exhibit non-peptidic characteristics such as resistance to boiling hydrochloric acid. See, Haupert, G. T., et al. "Hypothalamic Sodium-Transport Inhibitor Is A High-Affinity Reversible Inhibitor Of (Na,K)ATPase." Am J Physiol. 1984;247:F919-F924; Illescas, M., et al. "Partial Purification Of A Sodium Pump Inhibitor From Bovine Adenohypophysis. Its Comparison With The Natriuretic Factor Isolated From Hypothalamus." J Clin Exp Hypertension. 1988;A10:In press; Millett, J. A., et al. "Extraction And Characterization Of A Cytochemically Assayable Na/K-ATPase Inhibitor/Glucose-6-Phosphate Dehydrogenase Stimulator In The Hypothalamus And Plasma Of Man And The Rat." J Endocr. 1987;112:299-303.
Further compounding matters, two investigators utilizing plasma as their source of endogenous digitalis-like factors characterized their inhibitors as unsaturated fatty acids. See, Tamura, M., et al. "Identification Of Linoleic And Oleic Acids As Endogenous Na.sup.+ -K.sup.+ -ATPase Inhibitors From Acute Volume-expanded Hog Plasma. (Abs.)." J Biol Chem. 1985;260:9672-9677; Kelly, R. A., et al. "Identification Of NaK-ATPase Inhibitors In Human Plasma As Nonesterified Fatty Acids And Lysophospholipids." J Biol Chem. 1986;261 no.25:11704-11711.
In spite of the confusion regarding the identity and character of these compounds, that such endogenous sodium transport inhibitory factors may be implicated in several areas of human pathology has been established. Research has demonstrated that serum or urine from patients having essential hypertension contains sodium pump inhibitors. These inhibitory factors exhibit a variety of activities including inhibition of cellular active sodium fluxes, inhibition of Na.sup.+, K.sup.+ -ATPase, positive entropy of the heart, increase in the pressor response to noradrenaline, competition with radio-labelled cardiac glycosides for binding sites (typically .sup.3 H ouabain binding) as well as competition with radio-labelled antibodies raised against a cardiac glycoside.
It should be noted that the cardiac glycoside radioimmunoassay is the least reliable method for the detection of a sodium pump inhibitory factor because some compounds nonspecifically bind to common steroids such as aldosterone. See, Schreiber, V., et al. "Digoxin-Like Immunoreactivity Of Certain Steroid And Other Hormones." Physiol Biochem. 1981;30:569-571.
Numerous investigators have also shown that serum from patients with essential hypertension inhibits active sodium transport in normal leukocytes and lymphocytes. See, Oh, V., et al. "Reversible Inhibition Of Leucocyte Sodium Pumps By A Circulating Serum Factor In Essential Hypertension." Bri Med J. 1986;292:1551-1555; Gray, H. H., et al. "Effect Of Serum From Patient With Essential Hypertension On Sodium Transport In Normal Leukocytes." Clin Sci. 1986; 70:583-586; Moreth, K., et al. "Blood Pressure In Essential Hypertension Correlates With The Concentration Of A Circulating Inhibitor Of The Sodium Pump." Klin Wochens. 1986;64:239-244.
Poston, et al. incubated mixed, normal leukocytes with sera obtained from patents with essential hypertension and found that the depression of active sodium transport in the normal cells was equivalent to that in the cells of the patients. See, Poston, L., et al. "Evidence For A Circulating Sodium Transport Inhibitor In Essential Hypertension." Br Med J. 1981;282:1267-1269. Similarly, both Gray, et al. and Moreth, et al. found that the degree of abnormality conferred upon the normal cells correlated with the blood pressure of the patients from whom sera were obtained. Additionally, Oh and Taylor reported that incubation of serum from patients with essential hypertension with normal lymphocytes reduced the binding of radiolabeled ouabain in the cells.
Such results suggest the presence of a cardiac glucoside-like substance in the serum. Conversely, further contributing to the disparity of reported characteristics of such inhibitory factors, Boon, et al. could not demonstrate any effect of hypertensive serum on normal glucoside .sup.3 H ouabain binding after incubation. See, Boon, et al. "Cation Transport Functions In Vitro In Patients With Untreated Essential Hypertension: A Comparison Of Erythrocytes And Leukocytes." Clin Sci. 1985;68:511-515.
It should be appreciated that at present there is no work clearly suggesting that erythrocyte sodium fluxes are affected by a sodium pump inhibitor in essential hypertension. Poston, et al. failed to demonstrate any inhibitory effect of hypertensive serum on normal erythrocytes although the sodium content of the patients' cells was high. Similarly, Millar, et al. found high erythrocyte sodium in their patient group yet were unable to demonstrate any effect of their patients' serum on normal erythrocytes. Millar, J. A., et al. "Evidence Against A Circulating Ouabain-Like Transport Inhibitor As A Cause Of Increased Red Cell Sodium In Essential Hypertension." J Hipert. 1984;2:461-463.
However, inhibition of .sup.3 H ouabain binding after incubation of normal erythrocytes with a plasma extract from essential hypertension patients has been demonstrated. Cloix, et al. "Plasma Endogenous Sodium Pump Inhibitor In Essential Hypertension." J Hypert. 1983;1:11-14. However, though this inhibition correlated with the rate of urinary sodium excretion it did not correlate with blood pressure.
In spite of the foregoing, research has suggested that serum from patients with essential hypertension has cardiac glucoside-like properties and strongly supports a link between cellular sodium transport and venous tone in essential hypertension. For example, serum from patients with essential hypertension sensitizes the vascular response to catecholamines in a manner similar to the cardiac glycosides. Rabbit aortic strips incubated with plasma from patients with essential hypertension and subsequently stimulated with nonadrenaline produced a greater contraction than that evidenced in aortic strips exposed to normal plasma. See, Michelakis, A. M., et al. "Further Studies On The Existence Of A Sensitizing Factor To Pressor Agents in Hypertension." J Clin Endocr Metab. 1975;41:90-96.
Additionally, studies have directly demonstrated the inhibition of Na.sup.+, K.sup.+ -ATPase by serum from patients with essential hypertension. For example, deproteinized plasma from normotensive and hypertensive subjects was tested on Na.sup.+, K.sup.+ -ATPase isolated from dog kidney. The hypertensive plasma inhibited the enzyme to a degree correlated with the mean arterial pressure of the subject. See. Hamlyn, J. M., et al. "A circulating Inhibitor Of (Na,K)ATPase Associated With Essential Hypertension." Nature. 1982;300;650652. Similarly, it has been reported that plasma extract which inhibited .sup.3 H ouabain binding in erythrocytes also inhibited dog kidney Na.sup.+, K.sup.+ -ATPase activity. See, Cloix, J. F., et al. "Plasma Endogenous Sodium Pump Inhibitor In Essential Hypertension." J Hypert. 1983;1:11-14.
Indirect evidence of the existence of a sodium pump inhibitor in the serum of patients with essential hypertension has also been found in a cytochemical assay for the enzyme glucose 6-phosphate dehydrogenase (G6PDH). For example, G6PDH in guinea pig kidney cortical slices is stimulated by the inhibition of Na.sup.+, K.sup.+ -ATPase. Accordingly, the degree of G6PDH stimulation by serum is an index of the serum Na.sup.+, K.sup.+ -ATPase inhibitory activity. Moreover, serum from patients with essential hypertension stimulates cortical G6PDH to a degree correlating with the diastolic blood pressure of the patients. See, MacGregor, G. A., et al., "Evidence For A Raised Concentration Of A Circulating Sodium Transport Inhibitor In Essential Hypertension." Br Med J. 1981;283:1355-1357.
There are also reports evidencing an endogenous digoxin-like activity in serum from patients with essential hypertension. See, Fett, J. W., et al. "Isolation And Characterization Of Angiogenin And Angiogenic Protein From Human Carcinoma Cells." Biochem. 1985;24:5840. Fractionation studies of the concentrated deproteinized serum utilizing gel filtration suggests that several substances may be involved in this activity.
In spite of this evidence confirming that sodium transport is abnormal in essential hypertension patients and that this abnormality is due to a circulating sodium pump inhibitory factor or factors, whether such factors are involved in the genesis of essential hypertension remains to be conclusively proven. Moreover, the relationship of the previously discussed inhibitory factors to essential hypertension also remains to be conclusively proven.
Some researchers have suggested that essential hypertension is associated with a Na.sup.+, K.sup.+ -ATPase inhibitory factor or factors which may account for abnormalities of the sodium pump. See, Haddy, F. J., et al. "Role Of A Humoral Sodium-Potassium Pump Inhibitor In Experimental Low Renin Hypertension." Life Sci. 19:30:571-575; de Wardener, H. E., et al. "Dahl's Hypothesis That A Saluretic Substance May Be Responsible For A Sustained Rise In Arterial Pressure: Its Possible Role In Essential Hypertension." Kidney Int. 1980;18:1-9. These reporters also suggest that this concept explains the effects of dietary sodium intake in hypertension. See, Houston, M. C. "Sodium And Hypertension." Arch Intern Med. 1986;146:179-185.
These same researchers propose an underlying, genetically determined kidney defect resulting in inadequate sodium excretion on a high salt intake and subsequent expansion of the ECFV. It is believed that this underlying defect may be an abnormality of sodium hydrogen exchange. See. Mahnensmith, R. L., et al. "The Plasma Membrane Sodium-Hydrogen Exchanger And Its Role In Physiological And Pathophysiological Processes." Circ Res. 1985;57:773-788.
Though there is uncertainty regarding the connection between sodium pump inhibitory factors and essential hypertension, it is clear that volume expansion leads to a production of a circulating sodium pump inhibitory factor or factors. By reducing renal tubular sodium reabsorption such factors could lead to a correction of the ECFV. Further, inhibition of sodium transport in vascular smooth muscle cells could also lead to a rise in their calcium content which would produce vasoconstriction and resultant high blood pressure. See, Blaustein, M. P., et al. "Role Of A Natriuretic Factor In Essential Hypertension Hypothesis." Ann of Inter Med. 1983;98:785-792.
However, experiments directed to determining the vasoconstrictor effect of endogenous sodium pump inhibitory factors derived from volume-expanded or from hypertensive patients have not been performed to conclusively establish this connection. Nonetheless, it is clear from the foregoing discussion that the experimental evidence presently in existence strongly argues in favor of the existence of endogenous sodium pump inhibitors which are implicated in the mechanism of essential hypertension and other physiopathological processes. See, Shlevin, H. H., "(Na-K-)-ATPase Inhibitors For New Drug Discovery." Drug Develop Res. 1984;4:275-284.
These physiopathological processes also include the formation of blood vessels, an important process in both embryonic and postnatal life. A variety of angiogenic factors have been described in the art. See, Auerbach, R., Angiogenesis-inducing factors: a review. In: Pick, E., ed. Lymphokinines. Vol. IV. New York: Academic Press; 1981:69-:88. These factors include those produced by normal tissues such as epidermal growth factor and acidic fibroblast growth factor. See, Folkman, J., Angiogenesis:initiation and control. J. Cell Biol. 1982;104:212; Furcht, L. T., "Critical factors controlling angiogenesis: cell products cell matrix, and growth factors." Lab. Invest. 1986;55:505; Gospodarowidcz, D., Thakral, K. K., "Production of a corpus luteum angiogenic factor responsible for proliferation of capillaries and neovascularization of the corpus luteum". Proc. Nat'l Acad. Sci. 1978;75:847; Weiner, H. L., Weiner, L. H., Swain, J. L., "Tissue distribution and developmental expression of the messenger RNA encoding angiogenin". Science. 1987;237:280. Angiogenic factors have also been reported in neoplasms of mammals including humans. See, Fett, J. W., Strydom, D. J., Lobb, R. R., et al., "Isolation and characterization of angiogenin and angiogenic protein from human carcinoma cells". Biochem. 1985;24:5840 and Weiner, H. L., Weiner, L. H., Swain, J. L., "Tissue distribution and developmental expression of the messenger RNA encoding angiogenin". Science. 1987;237:280; Guillino, P. M., "Angiogenesis and oncogenesis". J. Nat'l Canc. Inst. 1978;61:639; Kumar, S., West, D., Daniel M., Hancock, A. S., Carr, T., "Human lung tumour cell line adapted to grow in serum-free medium secretes antiogenesis factor". Int'l J. Canc. 1983;32:461; Schor, A. M., Schor, S. L., "Tumor antiogensis". J. Pathol. 1983;141:385; Shing, Y., Folkman, J., Haudenschild, C., Lund, D., Crum, R., Klsagsbrun, M., "Angiogenesis is stimulated by a tumor-derived endothelial cell growth factor". J. Cell Biol. 1985;29:275; Stenzinger, W., Gruggen, J., Macher, E., Sorg., C., "Tumor angiogenic activity (TAA) production in vitro and growth in the nude mouse by human malignant melanoma". Eur. J. Canc. Clin. Oncol. 1983;19:649. It is believed that inhibition of angiogenesis may be due to Na.sup.+, K.sup.+ -ATPase inhibition because fibroblast growth factor binding to its specific receptor is dependent upon the Na.sup.+ gradient across the cell membrane and more than 70% of Na.sup.+ movement across the plasmatic membrane is based upon sodium pump activity.
Accordingly, it is a principle object of the present invention to disclose and claim a substantially pure endogenous Na.sup.+, K.sup.+ -ATPase inhibitory factor. It is a further object of the present invention to provide effective methodologies for isolating and characterizing this endogenous Na.sup.+, K.sup.+ -ATPase inhibitory factor in its substantially pure form. In this manner, the present invention overcomes the inability of the earlier researchers to isolate, purify and identify such compounds.
Moreover, it is an additional object of the present invention to provide a substantially pure Na.sup.+, K.sup.+ -ATPase inhibitory factor which is biologically active at physiological concentrations of potassium. Further, those skilled in the art will appreciate that the biological activity of the inhibitory factor of the present invention functions in a dose responsive manner making it particularly effective as a pharmaceutical compound for use in therapeutic methodologies.
Thus, it is a still further object of the present invention to provide such pharmaceutical compositions and associated methods for their use in treating essential hypertension in mammals as well as for treating cardiac malfunction. Similarly, it is also an object of the present invention to provide pharmaceutical compositions and methods for their use in regulating active sodium transport in mammals as well as functioning as diuretics. It is also an object of the present invention to provide pharmaceutical compositions and methods for their use in inhibiting and/or regulating angiogenesis.
It is also an object of the present invention to clarify the contradictory state of the art with respect to the characterization of endogenous sodium pump inhibitory factors.